Method of making a pH and amine test element

ABSTRACT

Tests for elevated pH and volatile amines in aqueous fluids are disclosed, including tests useful in the diagnosis of bacterial vaginosis and in other biological conditions. By using formulated indicators and indicators held in matrices that are permeable to gas but not to liquid, the tests provide clear and sharp transitions detectable by visual or machine-readable means rather than by subjective judgments such as small gradations in color or olfactory determinations. The tests lend themselves readily to iconic readouts of the test indications and to the inclusion of positive and negative controls.

This application is a division of and claims the benefit of U.S.application Ser. No. 08/819.790 filed Mar. 18, 1997 now U.S. Pat.5,897,834 which is a continuation of U.S. application Ser. No.08/689,758 filed Aug. 13, 1996, now U.S. Pat. No. 5,660,790 issued Aug.26, 1997, the disclosures of which are incorporated by reference.

FIELD OF THE INVENTION

This invention relates to test devices for clinical use, and inparticular to test devices for conditions characterized by an abnormalpH in an aqueous fluid, and for conditions characterized by the presenceof volatile amines. A particular area of interest for the use of thesetests is the diagnosis of vaginal diseases.

BACKGROUND OF THE INVENTION

An early study of bacterial vaginosis (BV) involved comparisons of thepH of vaginal fluids of women known to be suffering from BV with thoseknown to be free of the disease. Gardner, H.L., et al., Am. J. Obstet.Gynecol. 69:962 (1955). All of the BV positive women in the study weredetermined to have a vaginal fluid pH greater than 4.5, and 91% of thesewomen had a vaginal fluid pH greater than 5.0. Of the normal(disease-free) women in the test, 92% were found to have vaginal pHbetween 4.0 and 4.7. The conclusion drawn from the study was that avaginal pH equal to or greater than 5.0 in conjunction with otherclinical criteria was indicative of the presence of BV. Subsequentstudies culminating in a report by Amsel, R., et al., Am. J. Med.74:14-22 (1983), resulted in a reduction of the pH threshold for BV to4.5, and established the remaining criteria as vaginal fluidhomogeneity, the whiff test (treatment with alkali followed by anolfactory test to detect for an amine odor), and the presence of cluecells. These are commonly referred to as the Amsel clinical criteria forBV. The conclusion was based on a study group of 397 women in which 81%of BV positive women were found to have a pH greater than 4.5 while only23% of the normal women were found to have a vaginal fluid pH greaterthan 4.5.

Studies subsequent to the report by Amsel et al. have now adjusted thepH threshold to 4.7. One of these is the study of Holst, E., J. Clin.Microbiol. 28:2035-2039 (1990), in which 100% of the women diagnosed asBV positive by the Amsel criteria were reported to have vaginal fluid pHgreater than 4.7. Another is the study by Eschenbach. D. A. Am. J.Obstet. Gynecol. 158(4):819-828 (1988), in which all 257 women in thestudy group who had at least 20% clue cells were shown to have a vaginalfluid pH greater than or equal to 4.7, leading to the conclusion that athreshold value of 4.7 correlated best with the other clinical evidenceof BV. Krohn. M. A., et al., J. Clin. Microbiol. 27(6):1266-1271 (1989),also verified the correlation between the vaginal fluid pH threshold of4.7 and the presence of clue cells, and Holmes. K. K., and coworkersfurther confirmed the pH 4.7 threshold as an indicator of BV--Holmes, K.K., et al., eds., Sexually Transmitted Diseases, McGraw-Hill, New York(1990), Chapter 46:527-545 (Holmes. K. K., et al.), and Chapter47:547-559 (Hillier, S. L., et al.).

Colorimetric tests for elevated vaginal fluid pH have used nitrazineyellow as an indicator. Nitrazine yellow is a monoazo dye that is brightyellow at pH 6.0 and bright blue at pH 7.2, and has a grey-greenmidpoint at pH 6.6. In the range of interest for testing vaginal fluid,however, which is approximately 3.5 to 6.0, the change occurs in subtleprogressions of grey-green that are difficult to interpret.

pH thresholds are useful indicators in diagnosing a variety of otherbiological conditions, in both humans and animals, and a large number ofcolorimetric indicators are known and commercially available. Selectionof the appropriate indicator is not always a simple task, however, andthe choices are often limited, particularly when a specific color changeis desired and when stability of the indicator is a consideration.

Normal bovine milk, for example, has a pH of 6.5 to 6.8, and it has beenreported (N. Z. J. Sci. Technol. 27:258 (1945)) that bovine milk with apH greater than 6.8 may indicate the presence of bovine mastitis. Thedifficulties of detecting a pH deviation of such a small magnitude usingconventional paper indicator strips are discussed above. A studyspecifically directed to bovine mastitis was reported in J. Dairy Sci.68: 1263-1269 (1985). The purpose of the study was to determine thesuitability of using absorbent blotting paper impregnated with the pHindicator bromthymol blue to test the pH of bovine milk as a method ofdetecting subclinical bovine mastitis. Milk was added to theindicator-treated paper, and the color of the pH indicator spot wasscored on a scale of 1 to 4, where 1 (pale green) was assessed as normal(negative), and 2, 3 and 4 (increasing from moderate green to darkblue-green) were considered abnormal (positive). The pH of the milk wasalso determined electronically with a carefully calibrated pH meter. Thetest results illustrated the difficulty in defining accurately the colorof the test area: the predictive value of a positive colorimetric testranged from 49% to 52% (i.e., 51% to 48% of the results were falsepositives).

As expected, an increase in the test score was accompanied by anincrease in the severity of mastitis as defined by other diagnosticmeasures. However, in milk from animals with less severe mastitis. theconsiderable overlapping of results "highlighted the possible error ininterpretation of indicator scores. The wide variation of milk pH [asdetermined electronically] within each BTB [Brom Thymol Blue color]score showed that the indicator results were not closely related to pH."The investigators stressed the importance of using color comparatorswhich resemble as closely as possible the actual pH test method beingutilized. If the colorimetric milk pH test results were to beinterpreted immediately, it was important to use comparators that werealso wetted with milk. If the colorimetric pH test results were to bedetermined after the milk spots had dried, it was advantageous to usedry comparators.

Returning to bacterial vaginosis, the whiff test, which is one of theAmsel criteria, originated in a study by Pheifer, et al., N. Engl. J.Med. 298:1429-1434 (1978), that reported the presence of acharacteristic fishy amine odor upon the addition of 10% KOH to avaginal fluid specimen from a woman with BV. The odor is caused by thealkaline volatilization of amine salts found in the vaginal fluid ofwomen with BV. Unfortunately, the test is highly subjective, it exposesthe health care worker to potential biological hazards, and it isdisagreeable and vulnerable to error, since it is performed on amicroscope slide which, due to the transient nature of the amine odor,must be placed directly under the nose and sniffed immediately after theaddition of the KOH.

Alternatives to the whiff test are analytical procedures such as highvoltage electrophoresis (Chen, K. C .S., et al., J. Clin. Invest.63:828-835 (1979)), thin-layer chromatography (Chen, K. C. S., et al.,J. Infect. Dis. 145:337-345 (1982), and Sanderson, B. E., et al., Br. J.Vener. Dis. 59:302-305 (1983)), gas chromatography (Gravett, M. G., etal. Obstet. Gynecol. 67:229-237 (1986), and Dravenieks, A., et al., J.Pharma. Sc. 59:495-501 (1970)), and high-performance liquidchromatography (Cook, R. L., et al., J. Clin. Microbiol. 30:870-877(1992)). These procedures, although more accurate and reliable than thewhiff test, are expensive, time-consuming, and not suitable for on-sitetesting in a physician's office or clinic.

Clue cells, which constitute a further Amsel criterion, areindependently correlated with BV, and in the hands of a skilledmicroscopist are a very sensitive and specific indication of thisinfection. Clue cells are squamous vaginal epithelial cells found invaginal fluid when BV is present. The cells are covered with numerousbacteria, giving them a stippled or granular appearance, and theirborders are obscured or fuzzy because of the adherence of numerous rodsor cocci. According to standard clinical practice, a diagnosis of BV isestablished when at least 20% of the detectable epithelial cells areclue cells. Holmes, et al., Sexually Transmitted Diseases, 2d ed.,McGraw-Hill, Inc., New York, 1990.

Distinguishing between true clue cells in which the adherent bacteriacompletely obscure the edges of the cells and cells with simply a fewadherent bacteria requires training and experience. One source of erroris similarity in appearance between clue cells and trichomonads. whiteblood cells and other vaginal fluid components, frequently resulting inan incorrect identification of these cells as clue cells, and thereforefalse positive test results. Another is that clue cells when present arefrequently obscured by numerous vaginal fluid components causing theclinician to miss the clue cells completely or to quantify them atlevels below their actual level. This can result in a false negativetest result. Therefore, it would be highly desirable to have a distinctanaslyte that is accurately and conveniently monitored and whosepresence is correlated with clue cells.

SUMMARY OF THE INVENTION

The present invention resides in several discoveries, of value both bythemselves and in specific applications including their use in thediagnosis of vaginal infection.

One of these discoveries lies in a method for formulating a pH indicatorof the type that contains both an ionizable phenol group and anegatively charged group, and in pH indicators formulated in thismanner. According to this discovery, an indicator of this type can beimmobilized in a solid polymer matrix containing quaternary ammoniumgroups with two useful results:

(1) the indicator will remain immobilized in the matrix when wetted withan aqueous liquid sample, i.e., the indicator will resist bleeding outof the matrix or into adjacent areas of the matrix; and

(2) the transition point of the indicator, i.e., the pH at which avisible color change occurs, is lowered to a degree depending on thedensity of quaternary ammonium groups in the polymer and the ratio ofthe polymer concentration to the indicator concentration, and issharpened to cause the transition to occur over a narrower range of pH.Thus, indicators can be adjusted to undergo transitions at selected pHvalues, increasing the range of indicators suitable for any selectedtransition point and enabling one to select a preferred indicator andadapt it for a particular use.

A related discovery is a test device for analyzing an aqueous liquidsample (usually a biological specimen) for a pH equal to or greater thana critical point in the range of 4.6 to 4.8 (preferably about 4.7) by adetectable transition, in combination with a positive control that showsa similarly detectable transition at a lower pH, the two changes beingindependently visible on the test device. The positive control is usefulin assuring that the indicator is not malfunctioning for reasons such asmanufacturing error in the device, and that the device has been exposedto sufficient sample to produce a reading if the sample were indeedpositive. In preferred embodiments, the test device also contains anegative control, independently visible, that includes indicator whosechange is caused not by application of a sample but by the decompositionor deterioration of the indicator itself.

A further discovery is a test device for detecting salts of volatileamines in an aqueous liquid sample (again, usually a biologicalspecimen). This device contains a dry, solid gaseous amine-releasingsubstance in addition to an amine indicator retained in a matrix that isimpermeable to aqueous liquids. The gaseous amine-releasing substanceeliminates the need for the operator of the test to handle aqueousalkali. A related discovery is that this test when performed on aspecimen of vaginal fluid serves as a reliable indication of thepresence of clue cells in the vaginal fluid, avoiding the need tomicroscopically observe and count clue cells.

A still further discovery is a test device that combines the test for pHat or above the critical point quoted above and the test for salts ofvolatile amines in a single aqueous liquid sample, with independentvisual indications. This device is of particular utility in thediagnosis of bacterial vaginosis. A related discovery is that thisdevice can be used to test for two of the four tests in the Amselcriteria for bacterial vaginosis. A further related discovery is thatthe tests in this device can be used by themselves as a means fordiagnosing bacterial vaginosis, without the two additional criteria ofthe Amsel test.

As explained in detail below, one implementation of the concepts of thisinvention is a combination test device for BV in vaginal fluid thatshows a sharp visual transition at pH 4.7, and provides a reliablevisual indication of the presence of amines indicative of BV. Aparticularly preferred pH indicator is nitrazine yellow which, when incombination with quaternary ammonium groups, changes directly fromgreenish-yellow to blue over a narrow pH range of approximately 0.1 pHunits as the pH rises, the transition centering around pH 4.7. Theindependently visible positive control changes color at a lower pHencountered when any specimen of the fluid is applied, regardless ofwhether or not BV is present. The two transitions are independentlyvisible such that the occurrence of both indicates a pH of 4.7 orhigher, while the occurrence of only the lower-pH transition (thepositive control) indicates a pH below 4.7. The two transitions can bearranged in a geometric pattern that serves as an indication of theresult. A particularly preferred pattern is a pair of orthogonal barsforming a plus sign, the horizontal bar containing the indicator thatchanging color at low pH and the vertical bar containing the nitrazineyellow and quaternary ammonium groups. A specimen with a pH below 4.7thereby produces a minus sign by creating a color change in thehorizontal bar only, while a specimen with a pH above 4.7 produces aplus sign by creating color changes in both horizontal and verticalbars. The background area serves as the negative control.

The amine test differentiates between amines volatilized by alkali andthose that are not volatilized by alkali by incorporating solid alkaliaccessible to the specimen, an indicator accessible to a liquidspecimen, and an indicator accessible only to vapors emitted by thespecimen, in the same device. Thus, the specimen is first contacted withthe solid alkali, then applied to both indicators, one of which willundergo a color change regardless of the presence or absence of volatileamines, and the other a color change only in the presence of volatileamines. As in the pH test, the two indicators can be arranged in ageometric pattern, preferably one in the form of an icon that symbolizesthe result in a readily recognizable manner. Like the pH test, a pair oforthogonal bars can be used, with the indicator that is accessible toliquid residing in a horizontal bar (the positive control) and theindicator that is accessible only to vapor residing in a vertical barcrossing the horizontal bar. A specimen without volatile amines thusproduces a minus sign by causing a color change in the horizontal baronly, while a specimen with volatile amines produces a plus sign bycausing a color change in both horizontal and vertical bars. Thebackground field surrounding both bars serves as the negative control.Preferred test devices include both the pH test and the amine test inseparate areas of the device. A single specimen is applied to the twotest areas separately, preferably the pH test first and the amine testsecond.

These and other features and advantages of the invention and itspreferred embodiments will be more readily understood by thedescriptions that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plan view of each of several laminae in one example of a pHtest element in accordance with this invention, the laminae shownseparately in the order in which they are applied.

FIG. 2a is a plan view of a laminate formed from the laminae of FIG. 1.FIG. 2b is a cross section of the various laminae of FIG. 2a, takenalong the line B--B thereof.

FIG. 3 is a plan view of each of several laminae in one example of anamine test element in accordance with this invention, the laminae shownseparately in the order in which they are applied.

FIG. 4a is a plan view of a laminate formed from the laminae of FIG. 3.FIG. 4b is a cross section of the various laminae of FIG. 4a, takenalong the line B--B thereof.

FIG. 5a is a plan view of a test card incorporating both the pH testelement and the amine test element shown in the preceding figures.

FIG. 5b is an enlarged and exploded side view of the test card of FIG.5a.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

pH Formulations and Tests

pH indicators with ionizable phenol groups and negatively charged groupsare many and varied. Preferred negatively charged groups are sulfate andsulfonate groups. Examples of indicators with ionizable phenol groupsand negatively charged groups are:

acid blue 92 (anazolene sodium, CAS No. 3861-73-2)

acid blue 29 (CAS No. 5850-35-1)

acid alizarin violet N (CAS No. 2092-55-9)

bromophenol blue (3',3",5',5"-tetrabromophenolsulfonephthalein, CAS No.155-39-9)

bromochlorophenol blue(3',3"-dibromo-5',5"-dichlorophenolsulfonephthalein, CAS No.102185-52-4)

bromocresol green (3',3",5',5"-tetrabromo-m-cresolsulfonephthalein, CASNo. 76-60-8)

chlorophenol red (3',3"-dichlorophenolsulfonephthalein, CAS No.4430-204)

bromocresol purple (5',5"-dibromo-o-cresol sulfonephthalein, CAS No.115-40-2)

alizarin complexone dihydrate((3,4-dihydroxy-2-anthraquinolyl)methyliminodiacetic acid, CAS No.3952-78-1)

alizarin red S monohydrate(3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonic acid, sodium salt, CASNo. 130-22-3)

bromothymol blue (3',3"-dibromothymolsulfonephthalein, CAS No. 76-59-5)brilliant yellow (CAS No. 3051-11-4)

phenol red (phenolsulfonephthalein, CAS No. 34487-61-1)

cresol red (3',3"-dimethylphenolsulfonephthalein, CAS No. 1733-12-6)

m-cresol purple (2',2"-dimethylphenolsulfonephthalein, CAS No.2303-01-7)

thymol blue (2',2"-dimethyl-3',3"b-diisopropylphenolsulfonephthalein,CAS No. 76-61-9)

nitrazine yellow (2-(2,4-dinitrophenylazo)naphthol-3,6-disulfonic acid,disodium salt, CAS No. 5423-07-4)

All of these indicators are readily available from commercial suppliers.Preferred indicators are bromophenol blue, bromochlorophenol blue,bromocresol green, bromocresol purple, bromothymol blue, brilliantyellow, and nitrazine yellow.

The quaternary ammonium groups in the polymer can be any groups capableof asserting a positive charge sufficient to form an ionic attractionwith the negatively charged group(s) in the indicator. Preferredquaternary ammonium groups are lower alkyl ammonium groups in which thealkyl groups are C₁ -C₄ alkyl groups. Trimethylammonium groups areparticularly preferred. The quaternary ammonium groups are believed tofacilitate the deprotonization of the ionizable phenol, causing thedeprotonization and hence the color change to occur at a lower pH and tooccur less gradually (i.e., in a narrower transition zone) as the pHrises.

The quantity of quaternary ammonium groups in the polymer formulationcan vary widely, depending on how far the color transition point is tobe lowered and the degree of sharpening desired for the transition. Inmany applications of this invention, the amount of quaternary ammoniumgroups will be selected to lower the mid-range transition point of theindicator by about 1.0 to about 3.0 pH units, and preferably by about1.5 to about 2.5 pH units, with about 2.0 pH units preferred. The amountof quaternary ammonium groups can also be expressed in terms of thealkali value of the resulting polymer. This value represents themilligrams of KOH equivalent to the basicity of the quaternary ammoniumgroups contained in 1 g of the dry polymer. In most implementations ofthis invention, the alkali value will range from about 5 to about 50,with values ranging from about 15 to about 40 preferred.

In preferred embodiments, the quaternary ammonium groups are bound to apolymer that is porous (or otherwise water-permeable) and hydrophilicand thereby permeable to aqueous liquid specimens. The hydrophiliccharacter of the polymer is due either to the presence of the quaternaryammonium groups or to other groups on the polymer structure. The polymeritself can be any polymer which is inert relative to the components ofthe specimen being tested, and which is solid and can be formed into athin layer, coating or lamina. The polymer should be insoluble inaqueous media in general, and in vaginal fluid for those embodiments ofthe invention that are designed for testing vaginal fluid. Examples ofsuitable polymers are transparent polymers such as acrylic polymers,particularly copolymers of acrylic and methacrylic acid esters, anddiethylaminoethyl cellulose. Two presently preferred polymers that arereadily available from commercial suppliers are EUDRAGIT® RL PO andEUDRAGIT® RS PO Ammonio Methacrylate Copolymers, which are copolymers ofmethyl, ethyl and trimethylammonioethyl esters of acrylic andmethacrylic acids, as chloride salts. The quaternary ammonium groups arepresent on these polymers as trimethylammonioethyl esters ofmethacrylate units that constitute 10.4% (RL PO) or 5.6% (RS PO) byweight of the polymer. The alkali values are 28.1 and 15.2,respectively. These polymers are available from Rohm Tech Inc., Maiden,Mass., USA.

The use of nitrazine yellow as the indicator and thetrimethylammonioethyl esters of EUDRAGIT RL PO can serve as an example.The transition point of nitrazine yellow in the absence of quaternaryammonium groups is about pH 6.6. By varying the relative amounts ofnitrazine yellow and this particular polymer, one can lower thetransition point by a selected amount. Applying the polymer-indicatormixture as a liquid solution of which the polymer constitutes 25.0% byweight and the indicator constitutes varying amounts ranging from 0.1%to 0.6% by weight, the transition point of the indicator will be loweredto pH 4.7 at nitriazine yellow concentrations of 0.2% and 0.34%, to pH4.4 at 0.36%, to pH 4.3 at 0.4%, and to pH 4.2 at 0.6%.

In general, best results will often be obtained within certain ranges ofthe ratio of indicator to quaternary ammonium groups. For nitrazineyellow and EUDRAGIT RL PO, the ratio, expressed as the weight ratio ofKOH equivalent in the polymer to nitrazine yellow, has a preferred rangeof about 1.0 to about 6.0, and a more preferred range of about 1.5 toabout 3.5. For EUDRAGIT RL PO, a preferred weight ratio range of polymerto nitrazine yellow is about 50 to about 250, and more preferably about75 to about 125. In terms of a liquid solution of the same polymer andindicator to be applied to the foundation lamina, a preferred range ofthe polymer in the solution is about 12% to about 35%, and a preferredrange of the nitrazine yellow is about 0.05% to about 1.0%, all byweight.

In test devices in accordance with this invention, the polymer andindicator form a solid thin, and preferably homogeneous and transparent,water-permeable layer (that can be referred to as a pH indicator lamina)that remains solid and intact (i.e., does not dissolve or disintegrateinto powder) when saturated with aqueous liquids. Additional componentscan optionally be included in this pH indicator lamina, or in thecomposition from which it is formed, for a variety of purposes. Examplesof such components are a vehicle for spreading or applying the laminacomponents to a surface, drying agents, penetrants, agents to facilitatewetting of the polymer by the sample, and agents to facilitatedepositing or adhering the mixture on the surface of a solid support.Still other optional components and the reasons for including them willbe readily apparent to those skilled in the art. Typical of these addedcomponents are low molecular weight alcohols, penetrants, and surfaceactive agents. Sorbitol is one example of a suitable penetrant.

As suggested above, the pH indicator lamina can be formed over thesurface of a support substrate or foundation lamina, for stability ofthe lamina and ease of manufacture. A typical example is a polyethyleneterephthalate film such as MYLAR® (Du Pont de Nemours & Co., Wilmington,Del., USA), which can be coated with ethyl cellulose or a similarcoating to improve the adherence of the pH indicator lamina componentsto the solid support.

In preferred test devices in accordance with this invention, theindicator that has been modified to change color at the desiredtransition point will occupy one geometric region on the device, whilean indicator in another geometric region will change color at a pH lowerthan the first by at least about 0.7 pH unit. A color change in thissecond region when contacted with a liquid whose pH lies between thetransition points of the two regions will inform the user that theindicator in the second region is in functional condition, and thisinformation is logically extended to both indicators in the device. Thesecond region thus serves as a specimen-activated positive controllamina, i.e., one that would show a color change upon application of aspecimen regardless of whether the pH of the specimen was above or belowthe transition point of the test. The color change in the positivecontrol region would thus indicate that the indicator in the test region(i.e., the pH indicator lamina) would also show a color change if the pHwere high enough to exceed the transition point, and that sufficientsample was applied to wet the surface of the device. Using a pHindicator lamina with nitrazine yellow adjusted to a transition point ofpH 4.7 and vaginal fluid as an example, the positive control lamina willpreferably contain an indicator having a transition point at a pH thatis 0.7 or more pH units below 4.7, more preferably one whose transitionpoint lies at a pH of 4.0 or below, and most preferably at a pH of 3.5or below. It is particularly preferred that the color change be the sameor similar to the color change undergone by the indicator changing at pH4.7.

An example of an indicator that is useful for the positive control whenthe pH indicator lamina has a transition point of 4.7 is a mixture ofnitrazine yellow and bromocresol green. The weight ratio of nitrazineyellow and bromocresol green in such a mixture can vary and differentratios will produce different transition points. In general, however,best results will be obtained with a weight ratio (bromocresol green tonitrazine yellow) of about 0.05 to about 20.0, and preferably from about0.5 to about 5.0. This indicator or indicator combination can likewisebe immobilized in a water-permeable polymer, and the same polymerimpregnated with the pH 4.7 indicator can be used for the lower-pHindicator as well, although the invention is not restricted to devicesutilizing the same polymer in both locations. When the polymer andindicators are applied as a liquid solution, the preferred amounts ofthe indicators in the solution are from about 0.05% to about 1.0% byweight each.

The indicators (i.e., the pH indicator lamina and the positive controllamina) are preferably arranged on the test device so that the two colorchanges are independently visible, and it is further preferred that thedevice include a third indicator region that remains visible butprotected from the specimen so that application of the specimen itselfdoes not cause this indicator to change color. This non-changing regionthus serves as a negative background control, since any deviation fromits original color indicates that the indicator itself has decomposed orhas otherwise undergone a chemical conversion adverse to itseffectiveness in the test, or has a defect incorporated in it during themanufacturing stage, or that an inadequate amount of sample has beenapplied.

As indicated above, a preferred configuration of the indicators is twocrossed bars forming a plus sign. FIGS. 1, 2a, and 2b illustrate alaminate having this configuration. A foundation lamina 11 supports thepH indicator lamina 12 (whose transition point is pH 4.7) toward thelower surface of the device. An intermediate or liquid-channeling lamina13 is applied over the pH 4.7 indicator lamina, the liquid-channelinglamina being transparent and impermeable to liquid but having anelongated gap 14 in its center in the form of a vertical bar thatpermits the passage of liquid and forms the vertical bar of the plussign. The positive control lamina 15 (with the lower-pH indicator) isthen applied over the intermediate lamina in the form of a bar thatserves as the horizontal portion of the plus sign.

A specimen applied to the entire upper surface of the device thus wetsthe entire positive control lamina and passes through and around it tothe intermediate lamina which permits penetration only through the gap,thereby reaching the underlying pH 4.7 indicator lamina only on theregion 16 beneath the gap. Any color change in the pH 4.7 indicator isrestricted to the vertical bar 16 region under the gap. The remainder ofthe pH 4.7 indicator lamina 17 (the area surrounding the plus sign) isprotected from the specimen and thus does not change color. Vaginalfluid specimens that have pH less than 4.7 will produce a minus sign onthe device (only the positive control indicator bar 15 will be visible),while specimens with pH 4.7 or greater will produce a plus sign (bothbars 15 and 16 visible).

By using the most basic and fundamental concepts of this invention, pHtest devices are prepared that are capable of determining whether the pHof an aqueous liquid sample exceeds a selected threshold value by acolor change in a defined geometric region of the device, whileimmobilizing the indicator on the device so that the indicator does notmigrate when wetted by the sample, and while modifying the indicator sothat it undergoes a transition within a relatively sharp pH range. Thethreshold value is selected first by selection of the indicator, andsecond by adjustment of the transition point of the indicator by theincorporation of quaternary ammonium groups in the matrix. Aqueousliquids that can be tested by this device include aqueous solutions aswell as suspensions and emulsions in which the continuous phase is wateror an aqueous solution. Although aqueous liquids from any source can beused, the invention will be of primary interest in biological fluids,such as urine, saliva, blood, and vaginal fluid. In vaginal fluid, asindicated above, a prime example of the use of this invention is in thedetection of bacterial vaginosis. Vaginal fluid can also be tested forother purposes by this invention, such as for example for the pH change(a rise from 4.5 to 7.0 and higher) accompanying the rupture of theamniotic membrane. The invention will be of interest not only in testinghuman fluids, but also fluids from animals, such as livestock and pets.One example is the detection of mastitis in cattle by determiningwhether the pH of the milk has reached or surpassed a threshold value ofapproximately 6.8.

Amine Test

Like the pH test device of this invention, the amine test device, or theamine test portion of a test device that combines both tests, is alaminated structure. Individual laminae in the structure performdistinct and complementary functions, the laminae collectively coactingto provide an accurate and reproducible test whose results are readentirely by visual indications. Two key laminae are a solidgas-releasing lamina immediately accessible to the fluid specimen and agas-permeable indicator lamina that is permeable to gas and impermeableto the aqueous liquid specimen itself or to any of its components thatare not in gaseous form. The gas-releasing lamina is a solid lamina ofalkali that reacts with amine salts in the specimen and converts them tovolatile amines. The volatilized amines then penetrate the gas-permeableindicator lamina where they cause a color change in the indicator.

The terms "volatile amines" and "volatilized amines" include amines thatare only slightly volatile as well as those that are sufficientlyvolatile to escape into the atmosphere at substantial rates. Slightlyvolatile amines are those that form only a thin film of gas at theliquid surface without significant amounts diffusing into theatmosphere. This thin film of gas however is sufficient to penetrate thegas-permeable indicator lamina.

The choice of solid alkali for the gas-releasing lamina is not criticaland can vary. In general, alkali and alkaline earth metal aluminates,carbonates and hydroxides can be used. Best results will most often beachieved with the use of either sodium aluminate, sodium carbonate, ormagnesium hydroxide. Sodium aluminate is particularly preferred.

Permeability only to gas in the gas-permeable indicator lamina can beachieved in a variety of ways, depending on the nature of the sample. Aconvenient method for aqueous specimens is the use of a poroushydrophobic polymer. Suitable polymers are those that are solid,insoluble in aqueous fluids, particularly vaginal fluid, and readilyformed into a layer, coating or lamina that does not dissolve, disperseinto particulate form, or otherwise disintegrate upon contact with thesefluids. Examples of such polymers are ethyl cellulose, cellulose acetateand cellulose nitrate. Ethyl cellulose is particularly preferred.Alternatively, the indicator can reside in a hydrophilic water-permeablepolymer that is covered by a hydrophobic lamina.

Any indicator that changes color upon exposure to amines, and preferablyamines in a fluid specimen that would otherwise be acidic, may be used.Bromocresol green is one example, and may be used here as well as in thepH test. Other examples are bromophenol blue, bromocresol purple,bromochlorophenol blue, nitrazine yellow, and various other indicatorsamong those listed above.

The gas-releasing lamina and the gas-permeable indicator lamina arearranged in the test device such that contact between the alkali in thegas-releasing lamina and the indicator in the gas-permeable lamina doesnot occur in the absence of a fluid specimen. The arrangement is such topermit the user to first contact the gas-releasing lamina with thespecimen (preferably using a swab), and then contact the gas-permeableindicator lamina with the same specimen, so that gas present in thespecimen will penetrate the gas-indicator lamina.

The gas-permeable indicator lamina may thus be the lowermost lamina inthe device, furthest away from the surface to which the specimen isapplied, and the last lamina to be influenced by the specimen. Thislamina can optionally be applied over a foundation or support lamina, asin the pH indicator lamina. With the use of polymer such as ethylcellulose as the lamina matrix, however, the lamina is structurallystable and reliable in performance without the use of an underlyingfoundation lamina.

In addition to the arrangement of these two laminae relative to eachother, the generation of the volatile amines by the device and thepenetration of these amines to the indicator is enhanced by the use ofintervening laminae which can also serve to define geometrical regionswhere the color change will occur, plus the same types of information asprovided by the pH test.

As in the pH test, the color change in the amine test is preferablyarranged in such a manner that it causes a particular geometric indiciumto appear that is otherwise invisible. One means of achieving this is torestrict the access of the volatile amines to a defined geometricalregion in the gas-permeable indicator such that the remaining regions ofthe lamina are still visible. This can be achieved by applying agas-channeling lamina directly over the gas-permeable lamina, thegas-channeling lamina being lighttransmissive or transparent and made ofa gas-impermeable material except for an opening or passage within itsborders. The volatile amines are thereby channeled through the opening,and the only portion of the gas-permeable indicator lamina that shows acolor change is the area directly underneath the opening. Examples oflight-transmissive materials that are impermeable to gas are variouspolymers, particularly polyamides, polyacrylates, shellacs andvarnishes.

As further protection of the indicator in the gas-permeable lamina fromliquid amines, a lamina that is permeable to gas but impermeable toliquid can be placed directly over the gas-channeling lamina. Forconvenience, this protective lamina may be constructed of the samepolymer used in the gas-permeable indicator lamina. Here again, ethylcellulose is preferred.

A second indicator region can be included in the amine test device torespond to the presence of the specimen regardless of whether or notthere are volatile or volatilizable amines present in the specimen. Asin the pH test, this second indicator region serves as aspecimen-activated positive control, and can be applied as a furtherlamina designated a positive control lamina. The indicator in thislamina can be immobilized in a matrix that will permit the penetrationof the fluid sample, and the indicator itself can be any indicator thatwill change color upon application of the sample. The color change maybe due to the nonvolatile amines in the sample, or to the solid alkaliin the gas-releasing lamina as the alkali is drawn into the sample bythe applicator swab. The immobilizing matrix can be a hydrophilicpolymer, and prominent examples are the same polymers cited above aspreferred for use in the pH indicator lamina of the pH test. Theindicator can be the same indicator used in the gas-permeable indicatorlamina, although any indicator producing a color change upon contactwith the specimen can be used.

As in the pH test, the two indicators (i.e., the indicator accessibleonly by volatilized amines and the indicator in the positive controllamina which is accessible by both volatilized and nonvolatilized aminesas well as the alkalinized sample) are arranged so that the two colorchanges are independently visible. It is further preferred that thedevice include a third indicator region that remains visible butprotected from the specimen so that it does not change color under anycircumstances. This non-changing region thus serves as a negativebackground control for the same reasons as the negative control in thepH test.

Here as well, a preferred configuration of the indicators is two crossedbars forming a plus sign. Referring to FIGS. 3, 4a, and 4b, this can beachieved with the gas-permeable indicator lamina 21 as the lower lamina,and the clear gas-channeling lamina 22 applied directly over it. Anelongated gap 23 in the gas-channeling lamina divides it into twoseparated areas. The gap permits passage of the volatilized amines andforms the vertical bar of the plus sign. A protective liquid barrierlamina 24 (also clear) can also cover the gas-channeling lamina 22 aswell as the gap 23. The positive control lamina 25 (which permitspenetration by the liquid specimen) is then applied over the liquidbarrier lamina 24, the positive control lamina being in the shape of abar forming the horizontal portion of the plus sign. Finally, thegas-releasing lamina 26 is applied as a separate layer on the device,either over the protective liquid barrier 24 or outside of all of thelamina. The gas-releasing lamina in the arrangement shown in the drawingforms a ring around the horizontal bar of the positive control lamina,preferably not touching the bar. A small gap between the ring and thebar prevents a premature color change in the bar.

To use an amine test having the configuration described in the precedingparagraph, a swab moistened with vaginal fluid is wiped around thegas-releasing ring 26, then wiped across the entire circular area insidethe ring, thereby contacting the positive control horizontal bar 25 andthe regions above and below it. The horizontal bar 25 will change colorupon contact with any specimen, and any volatile amines released by thecontact of the swab with the gas-releasing ring will penetrate theliquid barrier lamina 24 and pass through the vertical bar gap 23 in thegas-channeling lamina to the gas-permeable indicator lamina 21. Thecolor change in the latter is restricted to the region of the verticalbar 27, while the remainder of the indicator in the gas-permeable lamina(the area 28 surrounding the plus sign) is protected from the specimenand thus does not change color. Vaginal fluid specimens that do notcontain volatilizable amines will thus produce a minus sign on thedevice (by causing a color change only in the horizontal bar 25), whilespecimens that do contain volatilizable amines will produce a plus sign(by causing color changes in both horizontal 25 and vertical 27 bars).

The indicators described above in connection with both the pH test andthe amine test are visual indicators, yielding a color change that isdetectable by the naked eye. As alternatives, this invention furthercontemplates machine-readable indicators, or those that becomedetectable upon excitation, such as fluorescence-readable indicators.

In preferred embodiments of the invention, the two tests are combined ona single test card 30 as shown in FIGS. 5a and 5b, FIG. 5b being anenlarged and exploded side view of FIG. 5a. The two tests will appear asseparate areas 31, 32 on one surface of the card. Each can be outlinedin a dark circle 33, 34 for enhanced visibility to the user. The darkcircle 34 surrounding the amine test area can be wider since it willcontain the gas-releasing lamina needed to perform the test. As shown inFIG. 5b, each of the test areas 31, 32 is formed of the laminates shownin the previous figures. The laminate of FIG. 2b is shown under testarea 31, and the laminate of FIG. 4b is shown under test area 32, withthe individual laminae identified by the same numerals used in FIGS. 2band 4b. The card can further contain such indicia as identifyinginformation and instructions for use. The upper surface 35 of the cardcan be covered with a protective sheet for protection during storage andshipping, but readily removable immediately prior to use. Application ofa single biological specimen to both test areas on the card is readilyaccomplished by a cotton-tipped stick or any swab capable of retaining asample and applying it to a surface. A typical test card in accordancewith this invention is approximately the size of a credit card, orapproximately 2 inches (5.1 cm) by 3 inches (7.6 cm) and the test areasare circular, with diameters of approximately 0.25 inch (0.635 cm).

The following examples are offered for purposes of illustration only. Inthese examples, test elements in accordance with the invention were usedhaving the configurations shown in the Figures. In the pH test element,the laminae were as follows, using the reference numbers of FIG. 1:

foundation lamina 11: Mylar support with ethyl cellulose coating appliedas a 15 weight percent solution of ethyl cellulose in n-propanol pHindicator lamina 12: EUDRAGIT RL PO and nitrazine yellow applied as asolution with the following composition: 25.0% EUDRAGIT RL PO, 0.34%nitrazine yellow, 8.6% sorbitol (wetting and permeability agent, 70%aqueous solution), 10.0% 2-ethoxy ethanol, 12.4% deionized water, 43.66%1-propanol (all by weight)

liquid-channeling barrier lamina 13: ethyl cellulose, applied as a 15%solution in 30% n-propanol (all by weight), containing a gap 14

positive control lamina 15: EUDRAGIT RL PO with nitrazine yellow andbromocresol green, applied as a solution with the following composition:22.8% n-propanol, 12.40% deionized water, 30.00% 2-ethoxyethanol, 8.60%sorbitol (70% solution in water), 25.0% EUDRAGIT RL PO, 0.17% nitrazineyellow, 0.30% bromocresol green, and 0.65% 2-sulfobenzoic acid anhydride(for moisture resistance) (all by weight)

In the amine test element, the laminae were as follows, using thereference numbers of FIG. 3:

gas-permeable indicator lamina 21: bromocresol green in ethyl cellulose,formed from a solution of the following composition: 1.8% bromocresolgreen, 12% ethyl cellulose, and 86.2% n-propanol (all by weight)

gas-channeling lamina 22: clear polyamide, applied as a solution of 20%polyamide resin, 50% n-propyl acetate and 30% n-propanol (all by weight)

liquid barrier 24: ethyl cellulose applied as a solution of 10 weightpercent ethyl cellulose in n-propyl alcohol

positive control lamina 25: EUDRAGIT RL PO and bromocresol green appliedas a solution with the following composition: 25.0% EUDRAGIT RL PO, 0.1%bromocresol green, 30.0% 2-ethoxy ethanol, 6.0% deionized water, 36.9%1-propanol, 2.0% 2-sulfobenzoic acid anhydride (all by weight)

gas-releasing lamina 26: sodium alumina applied as a solution of 28%sodium aluminate, 18% polyethylene, 3% maltodextrin, and 51% deionizedwater (all by weight).

To compare these test elements with elements and procedures of the priorart, two such procedures were used. One was the use of a COLORPHAST® pHtest strip, manufactured by EM Science, Gibbstown, N.J., USA, currentlysold for use by physicians. The other was the whiff test referred toabove in the Background of the Invention, and consisting of applying aportion of the specimen to a microscope slide, applying a drop of 10%KOH, and then performing an olfactory determination.

EXAMPLE 1

The pH test element in accordance with this invention was compared witha COLORPHAST pH test strip in performing pH tests on 607 vaginal fluidspecimens.

Of the 607 specimens, 298 were negative (pH<4.7) according to theCOLORPHAST test strip, and 258 of these were also negative according tothe test element of this invention. This represents 86.6% negativeagreement, with a standard deviation of 2.0% and a one-sided 95%confidence interval of 83.3%.

The specimens indicated by the COLORPHAST test strip as positive(pH≧4.7) numbered 309, and of these, 263 were positive as well accordingto the test element of this invention. This represents 85.1% positiveagreement, with a standard deviation of 2.0% and one-sided 95%confidence interval of 81.8%.

Overall agreement was 85.8%, standard deviation 1.4%, and confidenceinterval 83.5%.

EXAMPLE 2

In this example, the Amsel criteria were used to reconcile the specimensthat were not in agreement in the tests performed in Example 1. The fourAmsel criteria are elevated pH, the presence of amines by the whifftest, vaginal fluid homogeneity, and the presence of clue cells. Thetheory behind this test is that if the vaginal fluid pH was elevatedbecause of a vaginal infection, then at least two of the other threeAmsel criteria would also be expected to be positive, in keeping with aclinical diagnosis of bacterial vaginosis (BV).

The 86 discordant samples (where one pH test showed positive and theother negative) were subjected to the remaining three Amsel criteria. Ofthe 40 specimens that gave negative results by the COLORPHAST test stripand positive according to the invention test strip, only two wereclinically positive for BV by the Amsel criteria. Of the 46 specimensgiving positive results by the COLORPHAST test strip and negativeaccording to the invention test strip, 42 were clinically negative forBV by the Amsel criteria. This represents 98.5% positive agreement,88.8% negative agreement and 93.1% overall agreement, using Amselcriteria to reconcile discordant results.

EXAMPLE 3

This example compares the amine test element in accordance with thisinvention with the whiff test. Both tests were performed on a total of617 vaginal fluid specimens.

Of the total number of specimens tested, 196 gave a positive result bythe whiff test, and of these, 166 gave a positive result as well by thetest element of the invention, representing 84.7% positive agreement, astandard deviation of 2.5% and a one-sided 95% confidence interval of80.5%.

Of the total number tested, 421 gave a negative result by the whifftest, and of these, 385 also gave a negative result by the test elementof the invention, representing 91.4% negative agreement, a standarddeviation of 1.4% and a one-sided 95% confidence interval of 89.2%.

Overall agreement was 89.3%, standard agreement 1.2% and a one-sided 95%confidence interval of 87.3%.

EXAMPLE 4

Amsel criteria were again used to reconcile the specimens that were notin agreement in the amine tests. Here as well, if amines were detectedbecause of a vaginal infection, at least two of the other three Amselcriteria would be expected to be positive.

The 66 discordant amine test samples were subjected to the remainingthree Amsel criteria. Positive agreement rose to 91.2%, negativeagreement rose to 91.7%, and overall agreement to 91.6%.

EXAMPLE 5

This example compares the combined pH and amine tests of the presentinvention with the combined results of the COLORPHAST pH test strip andthe standard whiff test (the reference tests). According to this test,the result using the present invention was considered positive only whenboth the pH and amine tests gave positive results, and the result wasconsidered negative in all other cases, i.e., when either or both of thepH and amine tests gave a negative result. The reference tests weredesignated in the same manner--positive when both were positive, andnegative when any one or both were negative.

Of 604 vaginal fluid specimens, 184 specimens were positive according tothe reference tests, and 175 were positive according to the tests of theinvention. Likewise, 420 specimens were negative according to thereference test, and 429 were negative according to the invention tests.Disagreement occurred in only 45 cases. This represents 85.3% positiveagreement with standard deviation of 2.6% and a one-sided 95% confidenceinterval of 81.0%, 95.7% negative agreement with standard deviation of1.0% and a one-sided 95% confidence interval of 94.1%, and 92.5% overallagreement with standard deviation of 1.1% and a one-sided 95% confidenceinterval of 90.8%.

EXAMPLE 6

As with the individual test elements, discrepant results between thecombined tests of the invention and the combined reference tests werereconciled using the Amsel criteria to presumptively diagnose BV. Of the18 specimens that were negative according to the combined referencetests but positive according to the combined invention tests, only twowere clinically positive for BV according to the Amsel criteria. Of the27 specimens that were positive according to the reference tests butnegative according to the invention tests, ten were clinically negativefor BV according to the Amsel criteria. Thus, using the Amsel criteriato reconcile the discordant test results, positive agreement rose to90.3%, with standard deviation of 2.2% and a one-sided 95% confidenceinterval of 86.7%; negative agreement rose to 96.3%, with standarddeviation of 0.9% and a one-sided 95% confidence interval of 94.8%, andoverall agreement rose to 94.5% with standard deviation of 0.9% and aone-sided 95% confidence interval of 93.0%.

EXAMPLE 7

This example illustrates the use of the present invention in conjunctionwith the two additional Amsel criteria for the determination of thepresence or absence of bacterial vaginosis (BV). As indicated above,diagnoses using the Amsel method are based on four criteria: (1) a pHdetermination, (2) a whiff test for volatile amines, (3) an examinationof vaginal fluid homogeneity, and (4) a microscopic examination ofvaginal fluid for the presence of clue cells. According to traditionalmethods of evaluating the test results, women meeting at least three ofthe four criteria are considered to be BV positive. First, all women inthe study group were tested for all four criteria, using the COLORPHASTpH test strip for the pH test and the whiff test for the amines. Theanalysis was then repeated on the same group of women using the samefour criteria, but substituting the pH and amine tests of the presentinvention for the COLORPHAST and whiff tests.

The study group consisted of vaginal specimens from 604 women. Based onthe Amsel criteria using the COLORPHAST and whiff tests for the pH andamine determinations, 161 (26.6%) of the women were classified as BVpositive and 443 (73.4%) were classified as BV negative. Of the 161positives, 153 were also classified positive when the criteria wereretested using the pH and amine tests of the present invention. Of the443 negatives, 433 were also classified negative when the criteria wereretested using the pH and amine tests of the present invention. Thisrepresents 95.0% positive agreement, with 1.7% standard deviation andone-sided 95% confidence interval of 92.2%. Likewise, negative agreementwas 97.7%, with 0.7% standard deviation and one-sided 95% confidenceinterval of 96.6%. Overall agreement was 97.0%, with 0.7% standarddeviation and one-sided 95% confidence interval of 95.9%; and the kappacoefficient was 0.92, with 0.02 standard deviation and one-sided 95%confidence interval of 0.90.

The 18 discordant test results were reconciled by a Gram stain test asan independent means of diagnosing BV, where a Gram stain score of 7 orabove was considered BV positive. Of the 10 specimens that were BVnegative by the Amsel criteria when the COLORPHAST and whiff tests wereused and BV positive by the Amsel criteria when the pH and amine testsof the invention were used, 8 were clinically positive for BV by theGram stain test. Conversely, of the 8 specimens that were BV positive bythe Amsel criteria when the COLORPHAST and whiff tests were used and BVnegative when the tests of the invention were used, 1 was clinicallynegative by the Gram test. Thus, by reconciling discordant results withthe Gram stain test, positive agreement rose to 95.8%, negativeagreement to 99.5%, and overall agreement to 98.5% with kappacoefficient of 0.96, standard deviation of 0.01 and one-sided 95%confidence interval of 0.94.

EXAMPLE 8

This example illustrates the use of the pH and amine tests of thepresent invention by themselves as test criteria for BV. Comparisonswere made against the Amsel criteria (using the COLORPHAST strip for pHand the whiff test for amine), and the Gram stain test was again used toreconcile discordant results, where a Gram stain score of 7 or above wasconsidered BV positive. For the pH and amine tests of the presentinvention, the diagnosis was considered positive only when both testsgave positive results. When one or both gave negative results, thediagnosis was considered negative.

Of the 607 vaginal specimens tested, 162 were positive by the Amselcriteria, and of these 162, 139 were also positive by the pH and aminetests of the invention (85.8%. standard deviation 2.7%. one-sided 95%confidence interval 81.3%). Those testing negative by the Amsel criteriawere 445 in number, and of these 445, 408 (91.7%, standard deviation1.3%, one-sided 95% confidence interval 89.5%) were also negative by thepH and amine tests of the invention. This indicates a positivepredictive value of 79.0%, with standard deviation 3.1% and one-sided95% confidence interval 73.9%; a negative predictive value of 94.7%,with standard deviation 1.1% and one-sided 95% confidence interval92.9%; and an efficiency of 90.1%, with standard deviation 1.2% andone-sided 95% confidence interval 88.1%.

The 60 discordant test results were then reconciled with the Gram staintest. Of the 37 that were BV negative by the Amsel criteria and positiveby the invention tests, 24 were clinically positive by the Gram stain.Of the 23 that were BV positive by the Amsel criteria and negative bythe invention tests, 4 were clinically negative by the Gram stain.

Thus, by reconciling discordant test results with the Gram stain test,182 specimens were BV positive by the Gram-reconciled Amsel criteria,and of these, 163 were also positive by the two tests of the invention.Similarly, 425 were negative by the Gram-reconciled Amsel criteria, andof these, 412 were also negative by the two tests of the invention. Thisraised positive agreement to 89.6%, with standard deviation 2.3% andone-sided 95% confidence interval 85.8%; and it raised negativeagreement to 96.9%, with standard deviation 0.8% and one-sided 95%confidence interval 95.6%. These results indicate a positive predictivevalue of 92.6%, with standard deviation 2.0% and one-sided 95%confidence interval 89.4%; a negative predictive value of 95.6%, withstandard deviation 1.0% and one-sided 95% confidence interval 94.0%; andan efficiency of 94.7%, with standard deviation 0.9% and one-sided 95%confidence interval 93.2%.

EXAMPLE 9

This example illustrates the use of the amine test of the presentinvention as a test for clue cells. Comparisons were made againstmicroscopic detection of clue cells, and the Gram stain test was used toreconcile discordant results. A microscopic examination for clue cellswas considered positive when clue cells constituted more than 20% of thevaginal epithelial cells present in a vaginal fluid specimen. As in thepreceding examples, an amine test representing the present invention wasconsidered positive when the amine test element produced a blue plussign. Alternatively, the amine test representing the present inventionwas considered negative when the amine test element produced a blueminus sign.

Of the 625 vaginal fluid specimens tested, 167 were interpreted aspositive for clue cells by microscopy. Of these 167 positive clue cellspecimens, 135 were also positive by the amine test of the invention(80.8%). Specimens testing negative for clue cells by microscopy were458 in number. Of these 458, 388 were negative by the amine test of theinvention (84.7%). This indicates a positive agreement of 80.8%, anegative agreement of 84.7%, and an efficiency of 83.7%.

The 102 discordant test results were then reconciled with the Gram staintest. Of the 70 specimens that were clue cell negative by microscopy andpositive by the amine test of the present invention, 36 were clinicallypositive for BV by Gram stain analysis. Thus, by reconciling thediscordant 70 clue cell negative test results with the Gram stain, 194specimens were clue cell positive by Gram stain-reconciled microscopycriteria, and 171 of these 194 specimens were also positive by the aminetest of the invention.

Similarly, of the 32 specimens positive by the microscopy criteria andnegative by the amine test of the present invention, 9 specimens wereclinically negative for BV by Gram stain testing. Thus, by reconcilingthe discordant 32 clue cell positive test results with the Gram stain,431 specimens were clue cell negative by Gram stain-reconciledmicroscopy criteria, and 397 of these 431 specimens were also negativeby the amine test of the invention.

This raised the positive agreement to 88.1%, the negative agreement to92.1%, and the overall agreement to 90.9%.

The foregoing is offered primarily for purposes of illustration. It willbe readily apparent to those skilled in the art that the configurations,dimensions, reagents and other materials, procedural steps and otherparameters of this invention may be further modified or substituted invarious ways without departing from the spirit and scope of theinvention.

What is claimed is:
 1. A method for immobilizing a pH indicatorcontaining an ionizable phenol group and a negatively charged group in asolid matrix to prevent bleeding of said indicator when wetted with anaqueous liquid sample, and for providing said indicator with atransition region of a lower pH and a narrower range of pH relative tothat of said indicator when not so immobilized, said method comprisingforming a solid composition comprised of said indicator dispersed in apolymer to which quaternary ammonium groups are covalently bound, withsubstantially no covalent bonds between said indicator and said polymer.2. A method in accordance with claim 1 in which said pH indicator has amid-range transition point, and said quaternary ammonium groups arepresent in said polymer in an amount sufficient to lower said mid-rangetransition point by at least about 1.0 pH unit.
 3. A method inaccordance with claim 1 in which said pH indicator has a mid-rangetransition point, and said quaternary ammonium groups are present insaid polymer in an amount sufficient to lower said mid-range transitionpoint by from about 1.0 pH unit to about 3.0 pH units.
 4. A method inaccordance with claim 1 in which said pH indicator has a mid-rangetransition point, and said quaternary ammonium groups are present insaid polymer in an amount sufficient to lower said mid-range transitionpoint by from about 1.5 pH units to about 2.5 pH units.
 5. A method inaccordance with claim 1 in which said pH indicator has a mid-rangetransition point, and said quaternary ammonium groups are present in anamount sufficient to lower said mid-range transition point by about 2.0pH units.
 6. A method in accordance with claim 1 in which saidquaternary ammonium groups are present in an amount providing saidpolymer with an alkali value within the range of about 5 to about
 50. 7.A method in accordance with claim 1 in which said quaternary ammoniumgroups are present in an amount providing said polymer with an alkalivalue within the range of about 15 to about
 40. 8. A method inaccordance with claim 1 in which said quaternary ammonium groups aretrimethylanmmonium groups.
 9. A method in accordance with claim 1 inwhich said polymer is an acrylic polymer.
 10. A method in accordancewith claim 1 in which said polymer is a copolymer of acrylic andmethacrylic acid esters and said quaternary ammonium groups aretrimethylammonium groups.
 11. A method in accordance with claim 1 inwhich said negatively charged group of said pH indicator is a memberselected from the group consisting of sulfate and sulfonate groups. 12.A method in accordance with claim 1 in which said pH indicator is amember selected from the group consisting of bromophenol blue,bromochlorophenol blue, bromocresol blue, bromocresol purple, nitrazineyellow, bromothymol blue, and brilliant yellow.
 13. A method inaccordance with claim 1 in which said pH indicator is nitrazine yellow.